Developing genetic tools for a new model of infection and immunity

开发新感染和免疫模型的遗传工具

• 批准号: 8770409
• 负责人: Ioannis Eleftherianos
• 金额: $ 19.62万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-16 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8770409
• 关键词: Adolescent; Agriculture; Animal Model; Antigen Targeting; Area; Bacteria; Biological Models; Biology; Biomedical Research; Cadaver; Caenorhabditis elegans; Candidate Disease Gene; Cellular biology; Child; Chromosomes; Communicable Diseases; DNA; Development; Double-Stranded RNA; Drosophila genus; Drosophila melanogaster; Drug Targeting; Drug resistance; Elderly; Elements; Event; Exploratory/Developmental Grant; Food; Foundations; Future; Gene Expression; Gene Transfer Techniques; Generations; Genes; Genetic; Germ Lines; Health; Host Defense Mechanism; Human; Immune response; Immunity; Individual; Infection; Insecta; Intestines; Invaded; Investigation; Life; Life Cycle Stages; Microbe; Microinjections; Modeling; Molecular; Molecular Genetics; Morbidity - disease rate; Natural Immunity; Nature; Nematoda; Nematode infections; Organism; Parasites; Parasitic nematode; Pathogenicity; Pharmaceutical Preparations; Plague; Population; Positioning Attribute; Pregnant Women; Process; Production; Protocols documentation; RNA Interference; Recovery; Reporter; Reporter Genes; Research; Retrotransposon; Role; School-Age Population; Signal Transduction; Source; Staging; System; Techniques; Technology; Time; Transfection; Transgenes; Vaccine Antigen; Vaccines; Virulence Factors; World Bank; Xenorhabdus luminescens; base; disability-adjusted life years; exhaust; gain of function; gene function; immune function; improved; killings; knock-down; life history; method development; novel; pathogen; positional cloning; promoter; public health relevance; response; tool; vector

DESCRIPTION (provided by applicant): Parasitic nematode (PN) infections remain a major threat to human health worldwide, with more than 1 billion people infected. Children, pregnant women, and the elderly are particularly susceptible to morbidity from nematode infection. Control strategies are restricted to periodic de-worming of infected individuals, which is limited by rapid re-infection rates and the development of drug resistant worm populations. There are no vaccines available for PN infections in humans. Development of new drugs and vaccines will require a better understanding of PN biology, particularly the infective process and the host's immune response to infection. The requirement of an obligate host and the lack of good animal models have limited investigations into mechanisms by which PNs infect and usurp the host immune response. While the free living nematode Caenorhabditis elegans is an excellent model for nematode development, it is not a parasite. The insect parasitic nematode Heterorhabditis bacteriophora (Hb) offers potential as a tractable model of PN infection that has many of the advantages of C. elegans, including easy culture and manipulation of all life history stages. However, many of the powerful genetic tools, like transgenesis and RNA interference, have not been fully developed in Hb, restricting its use as a widespread model. We propose to develop these genetic tools in Hb with a focus on targeting the infective juvenile and early parasitic stages in order to investigate infection mechanisms. We will take two parallel but independent approaches to develop these tools that will provide a foundation for future investigation into the mechanism of PN infection. In Aim 1, we propose to develop a transfection protocol for Hb via germ-line microinjection of a piggybac retrotransposon-based integrating vector to introduce the reporter transgenes into Hb chromosomes. In Aim 2, we will develop a robust RNAi protocol for 2nd generation Hb hermaphrodites. By focusing on 2nd generation hermaphrodites we are ideally positioned to probe the biology of PN infection, as the progeny of this stage are the infective stage. Development of methods for gene knock down (reverse genetics) and transfection in this novel model nematode would represent a significant advance for PN research, and will allow investigation of PN gene function during infection for the first time.

描述（由申请方提供）：寄生线虫（PN）感染仍然是全球人类健康的主要威胁，感染人数超过10亿。儿童、孕妇和老年人特别容易因线虫感染而发病。控制策略仅限于对受感染个体进行定期驱虫，这受到快速再感染率和抗药性蠕虫种群发展的限制。目前尚无针对人类PN感染的疫苗。新药和疫苗的开发需要更好地了解PN生物学，特别是感染过程和宿主对感染的免疫反应。由于需要一个专性宿主和缺乏良好的动物模型，限制了对PN感染和篡夺宿主免疫应答机制的研究。虽然自由生活的线虫秀丽隐杆线虫是线虫发育的极好模型，但它不是寄生虫。昆虫寄生线虫异小杆线虫（Hb）具有C. elegans，包括容易的文化和操纵的所有生活史阶段。然而，许多强大的遗传工具，如转基因和RNA干扰，尚未在Hb中得到充分开发，限制了其作为广泛模型的使用。我们建议在Hb中开发这些遗传工具，重点针对感染性幼年和早期寄生阶段，以研究感染机制。我们将采取两种平行但独立的方法来开发这些工具，为将来研究PN感染的机制提供基础。在目标1中，我们提出了一种通过生殖细胞显微注射的piggybac逆转录转座子为基础的整合载体引入报告转基因到Hb染色体的Hb的转染协议。在目标2中，我们将为第二代Hb雌雄同体开发一种稳健的RNAi方案。通过关注第二代雌雄同体，我们理想地定位于探索PN感染的生物学，因为该阶段的后代是感染阶段。在这种新型线虫模型中开发基因敲除（反向遗传学）和转染方法将代表PN研究的重大进展，并将首次调查PN基因在感染过程中的功能。